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United States Patent | 6,071,744 |
Scholl ,   et al. | June 6, 2000 |
The present invention generally relates to the field of diagnostic virology. More particularly, the present invention is directed to methods and compositions useful for the detection and differentiation of viral particles and/or virions in specimens. The present invention also provides methods and compositions useful for evaluating the susceptibility of infectious viruses to antiviral agents.
Inventors: | Scholl; David R. (Athens, OH); Jollick; Joseph D. (Athens, OH) |
Assignee: | Diagnostic Hybrids, Inc. (Athens, OH); Ohio University (Athens, OH) |
Appl. No.: | 031495 |
Filed: | February 26, 1998 |
Current U.S. Class: | 435/325; 435/352; 435/366 |
Intern'l Class: | C12N 005/00; C12N 005/10 |
Field of Search: | 435/325,352,366 |
5418132 | May., 1995 | Olivo | 435/5. |
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TABLE 1 ______________________________________ # Specimens CPE .beta.-gal. Stain ______________________________________ 31 + + 62 - - 0 + - 3 - + ______________________________________
TABLE II ______________________________________ HSV-1 HSV-2 ______________________________________ 1. Distribution patterns Swirling, uniform dense Irregular, dispersed of blue stained cells. staining pre-dominate. pale to dense staining. 2. Morphologic and Small rounded, uniform Large, multi- color variation of fibroid, no syncytia. nucleate, many pale affected areas of the blue-green often monolayer. unstained syncytia. ______________________________________
TABLE III ______________________________________ .beta.-galactosidase Activity In HSV-1 And HSV-2 Infected Mixed Cell Monolayers As Measured By ONPG Hydrolysis.sup.1 OD.sub.405 /pfu .times. 10.sup.-3 % Activity HSV-2 Promoter/Reporter HSV-1 HSV-2 (HSV-1 = 100%) ______________________________________ ICP6/LacZ 0.209 0.019 8.99 ICP10/LacZ 0.049 0.008 -- ______________________________________ .sup.1 The ICP6 data presented here was recorded at 45 minutes in the ONP assay, while the ICP10 data were recorded after 135 minutes after incubation. The ICP10 values were so low (especially for HSV2 infection) that meaningful calculations could not be made. Because the ICP10 cell line produces less galactosidase per infected unit per unit time as compared to the ICP6 cell line, the data shown are not meant for #cell line comparisons, rather they are presented to illustrate the difference in galactosidase activity between HSV1 and 2 infected cells, regardless o the promoter controlling the reporter gene.
TABLE IV ______________________________________ Typing Of Clinical Specimens Using Two Different Assays Assay Employed .fwdarw. HSV Type Fluorescent Antibody Assay .dwnarw. Identified HSV-1 HSV-2 ______________________________________ ELVIS .TM. MCA HSV-1 29 0 HSV-2 0 27 ______________________________________
TABLE V ______________________________________ Day Set Tube # 1 2 3 4 5 6 7 ______________________________________ 1% 1. N PB 2. N PB 3. N PB 4. N PB NC 5. N N N N N N N NC 6. N N N N N N N 2% 1. N PB 2. N PB 3. N PB 4. N PB NC 5. N N N N N N N NC 6. N N N N N N N 3% 1. N PB 2. N PB 3. N PB 4. N N PB NC 5. N N N N N N N NC 6. N N N N N N N 4% 1. N PB 2. N PB 3. N PB 4. N PB NC 5. N N N N N N N NC 6. N N N N N N N ______________________________________ N = No CPE seen. P = CPE seen, no blue cells. PB = CPE seen, blue cells seen. NC = Negative control.
TABLE VI ______________________________________ Day Set Tube # 1 2 3 4 5 6 7 ______________________________________ 2% 1. PB (5 blue cells) 2. N PB (>10 blue cells) 3. N PB (>10 blue cells) 4. P (no blue) 5. PB (>10 blue cells) 6. N PB (>10 blue cells) 4% 1. PB (blue cells) 2. N PB (>10 blue cells) 3. N PB (>10 blue cells) 4. PB (>10 blue cells) 5. N PB (>10 blue cells) 6. N PB (>10 blue cells) 6% 1. PB (>10 blue cells) 2. PB (>10 blue cells) 3. PB (>10 blue cells) 4. N PB (>10 blue cells) 5. N PB (>10 blue cells) 6. PB (>10 blue cells) 8% 1. PB 2. N PB 3. N PB (many blue cells) 4. N PB 5. N PB 6. PB 10% 1. N PB 2. PB 3. N PB (many blue cells) 4. N N PB 5. N PB 6. PB ______________________________________ N = No CPE seen. P = CPE seen, no blue cells. BP = CPE seen, blue cells seen.
TABLE VII ______________________________________ HSV Type 1 Day Set Tube # 1 2 3 4 5 6 7 ______________________________________ 4% 1. N N PB 2. N PB 3. N PB 4. N N PB 5. N PB 6. N PB 6% 1. N PB 2. N N PB 3. N N PB 4. N PB 5. N PB 6. N PB 8% 1. N PB 2. N PB 3. N PB 4. N N PB 5. N N N PB 6. N PB 10% 1. N PB 2. N PB 3. N N PB 4. N PB 5. N PB 6. N PB 12% 1. N PB 2. N N PB 3. N N PB 4. N N PB 5. N PB 6. N PB 16% 1. N PB 2. N PB 3. N N PB 4. N PB 5. N PB 6. N N PB ______________________________________ N = No CPE seen. P = CPE seen, no blue cells. BP = CPE seen, blue cells seen.
TABLE VIII ______________________________________ HSV Type 2 Day Set Tube # 1 2 3 4 5 6 7 ______________________________________ 4% 1. N N PB 2. N PB 3. N N PB 4. N PB 5. N PB 6. N N PB 6% 1. N PB 2. N N PB 3. N N N PB 4. N PB 5. N N N N N N N 6. N PB 8% 1. N N PB 2. N N N N N N N 3. N N PB 4. N PB 5. N N N N N N N 6. N PB 10% 1. N PB 2. N N PB 3. N N PB 4. N N PB 5. N PB 6. N PB 12% 1. N PB 2. N PB 3. N N PB 4. N N PB 5. N N PB N N N N 6. N N N 16% 1. N PB 2. N PB 3. N N N N N N N 4. N N PB 5. N PB 6. N N PB ______________________________________ N = No CPE seen. P = CPE seen, no blue cells. BP = CPE seen, blue cells seen.
__________________________________________________________________________ # SEQUENCE LISTING - (1) GENERAL INFORMATION: - (iii) NUMBER OF SEQUENCES: 2 - (2) INFORMATION FOR SEQ ID NO:1: - (i) SEQUENCE CHARACTERISTICS: #pairs (A) LENGTH: 18 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - (ii) MOLECULE TYPE: DNA (genomic) - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: # 18 GC - (2) INFORMATION FOR SEQ ID NO:2: - (i) SEQUENCE CHARACTERISTICS: #pairs (A) LENGTH: 19 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - (ii) MOLECULE TYPE: DNA (genomic) - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: # 19 AGG __________________________________________________________________________